2008 plaque assay for human coronavirus nl63 using human colon carcinoma cells

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Open AcceMethodologyPlaque assay for human coronavirus NL63 using human colon carcinoma cellsPetra Herzog1,3, Christian Drosten*2 and Marcel A Müller2
Address: 1Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, D-20359 Hamburg, Germany, 2Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany and 3Qiagen Hamburg GmbH, Königstr. 4a, D-22767 Hamburg, Germany

Email: Petra Herzog - [email protected]; Christian Drosten* - [email protected]; Marcel A Müller - [email protected]

* Corresponding author

AbstractBackground: Coronaviruses cause a broad range of diseases in animals and humans. Humancoronavirus (hCoV) NL63 is associated with up to 10% of common colds. Viral plaque assays enablethe characterization of virus infectivity and allow for purifying virus stock solutions. They areessential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virusreplication and weak and diffuse cytopathogenic effects. It has not yet been possible to establishpracticable plaque assays for this important human pathogen.

Results: 12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Humancolon carcinoma cells (CaCo-2) replicated virus more than 100 fold more efficiently thancommonly used African green monkey kidney cells (LLC-MK2). CaCo-2 cells showedcytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlaysproved suitable for plaque assays. Best results were achieved with Avicel, which produced large andclear plaques from the 4th day of infection. The utility of plaque assays with agrose overlay wasdemonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL.

Conclusion: CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enablecytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agaroseoverlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will bebeneficial in antiviral screening, animal modelling of disease, and other experimental tasks.

BackgroundCoronaviruses are large enveloped plus-strand RNAviruses that are currently classified in three groups or pre-sumptive genera [1-3]. Group 1 is further divided into twophylogenetic clades exemplified by the transmissible gas-troenteritis virus (TGEV) and the porcine epidemic diar-rhoea virus (PEDV), respectively. The latter clade containstwo prototypic human coronaviruses (hCoV), termedhCoV-229E and -NL63 [4,5]. Like group 1, group 2 con-tains mammalian CoV. These include two human patho-

genic prototypes, termed hCoV-OC43 and -HKU1, severalimportant animal pathogens such as the bovine CoV andthe murine hepatitis virus, as well as the SARS-CoV [6-8].Group 3 contains foremostly avian CoV [9].

HCoV-229E and OC43 as well as the more recently iden-tified hCoV-HKU1 and – NL63 are major causes of com-mon colds in wintertime [10]. HCoV-NL63 was isolatedin African green monkey kidney cells (LLC-MK2) from aseven month old infant with bronchiolitis and conjuncti-

Published: 12 November 2008

Virology Journal 2008, 5:138 doi:10.1186/1743-422X-5-138

Received: 22 October 2008Accepted: 12 November 2008

This article is available from: http://www.virologyj.com/content/5/1/138

© 2008 Herzog et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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vitis [4]. In further investigations the virus was predomi-nantly detected in children with respiratory infections[11-14]. Up to 10% of children with respiratory diseaseyielded hCoV-NL63 [10,11,15-17].

Because of its relatively high prevalence hCoV-NL63 couldbecome an important model in screening for anti-corona-viral agents [12,18]. Several studies have suggested, e.g.,that hCoV protease inhibitors would be cross-reactiveamong different hCoV [19-21]. Antiviral screening relieson the detection of replicating virus in cell culture. For thisand other experimental tasks, plaque assays have provento be simple in application and efficacious in representingvirus viability.

Plaque assays make use of viscous overlays to cover cellsimmediately after infection, thus limiting virus spreadand restricting virus growth to foci of cells at the sites ofinitial infection. If virus contributes no or low cytopathiceffects to cells, these foci may be visualized by immunos-taining [22,23]. If virus induces strong cytopathogeniceffects (CPE), cells in plaques are lysed and plaques can bevisualized by staining of the residual intact cells.Cytopathogenic plaque assays are compatible with highthroughput screening [24,25] and facilitate plaque purifi-cation and cloning of virus. This in turn is helpful inobtaining virus stocks of optimized infectivity, e.g., bydecreasing the amount of defective interfering (di) parti-cles that accumulate during serial passaging of CoV [26].

Important technical achievements have been made instudying NL63 replication, including, most recently, thedevelopment of an infectious cDNA clone [27]. Still it is amajor obstacle that hCoV-NL63 replicates slowly and at

relatively low titres in all current cell cultures, such as LLC-MK2 and Vero-B4 cells [4,28,29]. Because the virus con-tributes very weak and diffuse CPE to these cells, there isno cytopathic plaque assay available for non-recombinantvirus [28].

Although hCoV-NL63 seems to replicate in the upper andlower airways, there are many CoV that predominantlyinfect the enteric tract, such as TGEV, PEDV, the felineenteric CoV, and the bovine coronavirus [30,31]. SARS-CoV was detected in faecal swabs from SARS patients [32].SARS-CoV was shown to replicate in colon carcinomacells (CaCo-2) [33] that are routinely used for growingentero- and adeno-, and astroviruses [34]. Interestingly,SARS-CoV and hCoV-NL63 were shown to use the samereceptor for virus entry, the angiotensin convertingenzyme 2 (ACE2) [35].

We show here that CaCo-2 cells are highly susceptible forhCoV-NL63 infections and that virus propagation in thesecells is much more efficient than in LLC-MK2 cells. By test-ing different overlays and assay formats we developedcytopathogenic NL63 plaque assays that can be used foranalytical and preparative purposes.

Results and discussionSusceptibility of different cell lines to hCoV-NL63 and cytopathogenic effectsLLC-MK2 and Vero cells do not cause clear CPE on infec-tion with hCoV-NL63. Because this virus uses the samereceptor as the SARS-CoV, 12 different cell cultures sus-ceptible to SARS-CoV infection were tested for susceptibil-ity to hCoV-NL63 [34,36,37] (Table 1). Cells in six-wellplates were infected with 10e4 plaque-forming units of

Table 1: Comparison of hCoV-NL63 replication by real time RT-PCR using different cell cultures

Designation* Day 0 [copies/μL] Day 7 [copies/μL] Amplification factor Cytopathogenic effect (CPE)

Vero E6 6.94e3 3.05e7 4.39e3 NoneVero FM 1.78e4 4.51e9 2.54e5 NoneCaCo-2 3.55e3 1.25e10 3.54e6 round and detached, dead cells with cell debris in

supernatant, strong effectCalu1 2.61e4 5.33e6 2.04e2 NoneCalu6 7.95e3 5.00e5 6.29e1 NonePOEK 8.11e4 3.03e5 3.74e0 NonePK13 2.66e2 7.78e5 2.93e3 None293lp 3.67e3 3.09e7 8.42e3 NoneFeA 1.45e4 5.83e5 4.03e1 NoneRD 3.14e5 1.57e4 4.99e-2 NonePS 1.20e4 1.44e6 1.19e2 NoneLLC-MK2 4.00e3 2.65e6 6.62e2 round and detached, weak effect

* Vero E6 rhesus kidney cells (ATCC CRL-1586), Vero FM rhesus kidney cells (ATCC CCL-81), CaCo-2 human colon carcinoma (ATCC HTB-37), Calu 1 human lung carcinoma (ICLC HTL95002), Calu 6 human lung carcinoma (ICLC HTL97003), POEK porcine foetal kidney (cell culture collection of the Robert Koch-Institute (RKI), Berlin, Germany), PK13 porcine kidney (cell culture collection of the Bernhard-Nocht-Institute (BNI), Hamburg, Germany), 293 human embryonic kidney (ATCC CRL-1573), FEA feline embryonic fibroblast (kindly provided by Dr. Marcel Asper, NewLab Inc., Cologne), RD human rhabdomyosarcoma cells (RKI), PS porcine kidney cells (RKI), and LLC-MK2 African green monkey kidney cells (ATTC CCL-7, kindly provided by Lia van der Hoek, Academic Medical Center Amsterdam, The Netherlands).



hCoV-NL63 virus stock LLC-MK2 NP. RNA concentra-tions in supernatants were measured short after virusadsorption (i.e., in fresh medium after washing off of theinfection supernatant), and 7 days later (Table 1). Increaseof virus RNA was less than 1000-fold in seven of 12 cul-tures. Interestingly, this included LLC-MK2, the prototypecell culture for NL63. In spite of a low amplification factorthese cells showed the usual weak CPE that is typicallyobserved when infected with hCoV-NL63.

Vero cells seemed to support virus growth efficiently butproduced no CPE. Interestingly, there was a remarkabledifference between Vero E6 and Vero FM cells (Table 1).In our hands these cells also showed differences in growthof SARS-CoV. Vero FM consistently showed more pro-nounced CPE than Vero E6 but there were no significantdifferences in RNA amplification (not shown).

CaCo-2 cells amplified virus RNA most efficiently, andshowed a clearly visible CPE starting from day 4 afterinfection. Cells became rounded, detached from the sur-face, and showed morphological signs of cell death (Fig-ure 1).

For confirmation of differential replication efficiencies,CaCo-2 and LLC-MK2 cells were infected in parallel. Bothcell lines were seeded in 25 cm2 flasks, and infected atmultiplicities of infection of 0.005. Samples of superna-tants were taken daily from day 0 to 7 and analyzed by realtime RT-PCR. As shown in Figure 2, CaCo-2 cells repli-cated virus more efficiently than LLC-MK2. From day 3

onward, RNA concentrations were more than 100 foldhigher in CaCo-2 cells. Because of the clear CPE observedin CaCo-2 cells, these cells were tested for their utility in acytopathogenic plaque assay.

Comparison of different overlaysThree overlay techniques commonly used for plaqueassays were tested for their suitability [23]. CaCo-2 cellswere infected in 6-well plates with hCoV-NL63. After onehour, supernatants were removed, cells washed with PBS,and overlaid as follows.

For CMC overlays, 1 mL fresh DMEM was added to eachwell. Subsequently 1 mL of 1.6% CMC solution wasslowly added per well. Agarose overlays (1% final concen-tration) were prepared by melting 2% agarose at 70°C,cooling it in a water bath to 42°C, and mixing it immedi-ately before application with an equal volume of 2 ×DMEM stored at room temperature. Two mL of the mix-ture were carefully applied to each well. Avicel overlayswere made by mixing 2.4% Avicel solution with an equalvolume of 2 × DMEM. 2 mL of the mixture were immedi-ately added to each well.

Plaque assays were incubated without disturbing at 37°Cand 5% CO2. Overlays were removed on day five and cellswere fixed with a solution of 4% formaldehyde in PBS.After 30 min the formaldehyde solution was removed,cells were washed twice with PBS, and stained with a 0.2%crystal violet solution. As shown in Figure 3, plaques were

Cytopathogenic effect of hCoV-NL63 on human colon carcinoma cells (CaCo-2)Figure 1Cytopathogenic effect of hCoV-NL63 on human colon carcinoma cells (CaCo-2). CaCo-2 cells 5 days after infec-tion with hCoV-NL63 at an multiplicity of infection of 0.1 (agarose overlay technique). A, mock-infection; B, infection. Photo-graphs were taken at 40-fold magnification; bars represent 20 μm.



visible with all three overlays, but staining was clearestwith Avicel.

Incubation timesHCoV-NL63 culture with LLC-MK2 cells takes more than7 days until first signs of weak CPE become visible. Inorder to test whether incubation times could be reducedwith CaCo-2 cells, five plaque assays on virus dilutionseries were done with Avicel overlays and terminated byfixation after 1, 2, 3, 4, and 5 days, respectively. On days1 and 2, no plaques were visible (not shown). Termina-tion at day 3 yielded plaques only at high virus concentra-tion (Figure 4). From day 4 onward, plaques were visiblein the lowest detectable virus concentration. Plaques onday 5 were larger, but did not increase in number.

Plaque preparationWork with hCoV-NL63 is complicated by low infectioustiters in virus stock solutions. In order to obtain more

infectious virus solutions, our standard virus stock LLC-MK2 NP (see Materials and Methods section) was plaque-purified using the agarose overlay. Because life staining ofcells with neutral red solution was not successful onCaCo-2 cells (not shown), we used an alternative tech-nique of plaque preparation.

Limiting dilution infections were done on 6-well plates.After 5 days, cytopathic foci were identified by scanningthrough the wells with an inverted microscope at lowmagnification, lighting through the clear agarose overlay.The positions of CPE foci were marked with a permanentmarker (it was helpful to turn up the microscope light forthis). The agarose overlay was penetrated with a pipetteand 10 to 20 μl of fluid was aspirated underneath theoverlay. This fluid was resuspended in 100 μl of Opti Proserum-free medium, which served as the starting solutionfor a new limiting dilution infection series in the next 6-well plate plaque assay. Three rounds of purification were

Growth kinetics of hCoV-NL63 on LLC-MK2 and CaCo-2 cellsFigure 2Growth kinetics of hCoV-NL63 on LLC-MK2 and CaCo-2 cells. 25 cm2 flasks of LLC-MK2 or CaCo-2 cells were infected at multiplicities of infection of 0.005 for 1 h, washed twice with PBS, and subsequently supplied with 10 mL DMEM. Samples were taken daily from day 0 to 7 (except day 4) and analyzed by real time RT-PCR. Error bars indicate ranges of three independent experiments.



done. After the last round, aspirated fluid was inoculatedin 5 mL of Opti Pro serum-free medium, which was thenoverlaid on confluent CaCo-2 cells in a 25 cm2 flask forinfection. After infection for one hour and washing, 5 mLDMEM were added and flasks were incubated at 37°C,5% CO2 for four days. Stocks were harvested and stored asdescribed for the original LLC-MK2 stock in the Materialsand Methods section. The purified virus is hereafterreferred to as CaCo-2 PP (for plaque-purified).

To compare the infectivity of the plaque-purified viruswith the original LLC-MK2 virus stock (see Materials andMethods section), viral titres were determined by Avicelplaque assay as shown in Figure 5. CaCo-2 PP was about10-fold more infectious than LLC-MK2 NP. Plaque assayswere repeated three times (not shown). Mean titres weredetermined to be 1.4 × 10e6 PFU/mL and 1.3 × 10e5 PFU/mL, respectively, for CaCo-2 PP and LLC-MK2 NP. Abso-lute quantification of virus RNA by real-time RT-PCRyielded 4.8 × 10e11 RNA copies/mL for CaCo-2 PP and5.3 × 10e10 copies/mL for LLC-MK2 NP.

It was interesting to note that both virus stocks had ratherhigh RNA concentrations as opposed to their infectivities.PFU/RNA ratios were 2.92 × 10e-6 for CaCo-2 PP and2.45 × 10e-6 for LLC-MK2 NP. This high excess of RNAover infectious units might be attributable to the virusharvesting procedure, possibly releasing nonpackagedRNA along with virus particles during freeze-thawing.Because PFU/RNA ratios were very similar for both stocks,it appeared unlikely that elimination of defective interfer-ing particles had contributed the gain of infectivity. It willbe interesting in future studies to see whether hCoV-NL63might have adapted to CaCo-2 cells during plaque purifi-cation.

ConclusionCaCo-2 cells seem to support hCoV-NL63 replication sig-nificantly better than hitherto used culture cells. Theirapplication for a cytopathogenic plaque assay facilitatesquantification of infectivity and enables studies usingplaque morphology. Short incubation time of 4 days iscompatible with high-throughput applications such asdrug screening. The use of Avicel as an overlay is favoura-ble for plaque quantification, whereas agarose overlaysare preferred for plaque purification. Virus stock ofincreased infectivity will be beneficial for antiviral screen-ing, animal modelling of disease, and other experimentaltasks.

MethodsCell culturesAll cells were cultivated in DMEM (Dulbecco's ModifiedEagles Medium) (PAA, Cölbe, Germany) with 4.5 g/L Glu-cose (PAA), supplemented with 10% Foetal Bovine Serum"GOLD" (PAA), 1% Penicillin/Streptomycin 100 × con-centrate (Penicillin 10000 U/mL, Streptomycin 10 mg/mL) (PAA), 1% L-Glutamine 200 mM, 1% Sodium Pyru-vate 100 mM (PAA), 1% MEM nonessential amino acids(NEAA) 100 × concentrate (PAA). Utilized cell cultures areidentified in Table 1. For passaging, cells were detachedusing trypsin-EDTA (PAA), except CaCo-2 cells. Thesewere routinely subcultured by scraping and pipetting formechanical re-suspension.

Plaque assay for hCoV-NL63 on CaCo-2 cells using different overlaysFigure 3Plaque assay for hCoV-NL63 on CaCo-2 cells using different overlays. HCoV-NL63 was serially diluted on CaCo-2 cells (10e-1 until 10e-5). After 1 h of virus adsorb-tion different overlays were added. After 5 days cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet solution. A) carboxymethyl-cellulose; B) agarose; C) Avicel.




Plaque assays with different incubation timesFigure 4Plaque assays with different incubation times. Plaque assays were performed with Avicel overlay and incubated for 3, 4, and 5 days, respectively. The dilution factor of LLC-MK2 NP virus stock used for infection is shown on the bottom.



Effect of plaque purificationFigure 5Effect of plaque purification. A, plaque assay with Avicel overlay on purified virus stock CaCo-2 PP. B, plaque assay on non-purified virus stock LLC-MK2 NP. C, viral RNA copies per mL of supernatant (left) and plaque forming units per mL of super-natant (right) for CaCo-2 PP and LLC-MK2 NP virus stocks (log scale). Error bars show ranges of three independent experi-ments.


HCoV-NL63 virus stock solutionAn eighth passage virus stock of hCoV-NL63 was kindlyprovided by Lia van der Hoek, AMC Amsterdam. It wasgrown in LLC-MK2 cells in limiting dilution series, recov-ering it three times from the last well of a dilution seriesstill showing diffuse CPE. Subconfluent LLC-MK2 monol-ayers were infected in 75 cm2 flasks with virus supernatantfrom the last round of limiting dilution culture at a ratioof 1:100 (200 μl virus supernatant in 20 mL of freshmedium). This concentration was the highest virus dilu-tion still infectious in this culture format. The flasks wereincubated at 37°C, 5% CO2, and harvested on day four.For harvesting, flasks were frozen at -70°C and thawed.Cells and supernatant were centrifuged for 10 min at 5000rpm. Cleared supernatant was aliquoted and stored at -70°C. This virus stock is hereafter referred to as LLC-MK2NP (for non-purified).

Infection of cellsCells were seeded in 6-well plates at approximately 4 ×10e5 cells per well and incubated until the monolayer was70–80% confluent. CaCo-2 cells were grown to 100%confluence. Prior to infection cells were washed with 1 ×phosphate buffered saline (PBS). Virus inoculum in 900μL GIBCO Opti Pro serum free medium (Invitrogen, Karl-sruhe, Germany) plus 1% Penicillin/Streptomycin (PAA)and 1% L-Glutamine (PAA) was added to each well. Inoc-ulum was removed after one hour of incubation. Cellswere washed twice with 1 × PBS and supplemented with 2mL DMEM per well.

RNA extraction and real time RT-PCRViral RNA was extracted from cell culture supernatantwith the QIAamp Viral RNA mini Kit (QIAGEN, Hilden,Germany). Real time RT-PCR for hCoV-NL63 with abso-lute virus RNA quantification was performed as describedpreviously [38].

Media and overlays for plaque assaysA 2.4% (w/v) suspension of Avicel RC-581 (FCM BioPol-ymer, Brussels, Belgium) was prepared in distilled waterand autoclaved (20 min 121°C)[23]. A 2% (w/v) suspen-sion of agarose (Plaque Agarose, Biozym, Hessisch Olden-dorf, Germany) was prepared in distilled water andautoclaved. A 1.6% carboxymethyl cellulose (CMC) solu-tion was prepared by autoclaving CMC powder (BDH,Poole, UK) with a magnetic stirrer. Autoclaved powderwas hydrated in DMEM at 1.6% (w/v) and stirred over-night until homogenous.

Double concentrated Dulbecco's modified Eagle medium(DMEM) was prepared by mixing DMEM (PAA) with 9.48g/L DMEM Powder (Biochrom, Berlin, Germany), supple-mented with 20% Foetal Bovine Serum "GOLD" (PAA),2% Penicillin/Streptomycin 100 × concentrate (Penicillin

10000 Units/mL, Streptomycin 10 mg/mL) (PAA), 2% L-Glutamine 200 mM, 2% Sodium Pyruvate 100 mM(PAA), 2% MEM NEAA 100 × concentrate (PAA). Mediumwas sterilized by filtration.

Competing interestsThe authors declare that they have no competing interests.

Authors' contributionsPH performed the experiments and wrote the manuscript.CD coordinated the experiments and wrote the manu-script. MAM performed the experiments and wrote themanuscript.

AcknowledgementsThis study was supported by the German Ministry of Education and Research (Project Code "Ökologie und Pathogenese von SARS"), and the European Commission (contract SSPE-CT-2005-022639).

References1. Masters PS: The molecular biology of coronaviruses. Adv Virus

Res 2006, 66:193-292.2. Gorbalenya AE, Snijder EJ, Spaan WJ: Severe acute respiratory

syndrome coronavirus phylogeny: toward consensus. J Virol2004, 78:7863-7866.

3. Gonzalez JM, Gomez-Puertas P, Cavanagh D, Gorbalenya AE,Enjuanes L: A comparative sequence analysis to revise the cur-rent taxonomy of the family Coronaviridae. Arch Virol 2003,148:2207-2235.

4. Hoek L van der, Pyrc K, Jebbink MF, Vermeulen-Oost W, BerkhoutRJ, Wolthers KC, Wertheim-van Dillen PM, Kaandorp J, Spaargaren J,Berkhout B: Identification of a new human coronavirus. NatMed 2004, 10:368-373.

5. Hamre D, Procknow JJ: A new virus isolated from the humanrespiratory tract. Proc Soc Exp Biol Med 1966, 121:190-193.

6. Pyrc K, Berkhout B, Hoek L van der: Identification of new humancoronaviruses. Expert Rev Anti Infect Ther 2007, 5:245-253.

7. Kahn JS: The widening scope of coronaviruses. Curr Opin Pediatr2006, 18:42-47.

8. Woo PC, Lau SK, Chu CM, Chan KH, Tsoi HW, Huang Y, Wong BH,Poon RW, Cai JJ, Luk WK, et al.: Characterization and completegenome sequence of a novel coronavirus, coronavirusHKU1, from patients with pneumonia. J Virol 2005, 79:884-895.

9. Cavanagh D: Coronaviruses in poultry and other birds. AvianPathol 2005, 34:439-448.

10. Pyrc K, Berkhout B, Hoek L van der: The novel human coronavi-ruses NL63 and HKU1. J Virol 2007, 81:3051-3057.

11. Dijkman R, Jebbink MF, El Idrissi NB, Pyrc K, Muller MA, Kuijpers TW,Zaaijer HL, Hoek L van der: Human coronavirus NL63 and 229Eseroconversion in children. J Clin Microbiol 2008, 46:2368-2373.

12. Pyrc K, Berkhout B, Hoek L van der: Antiviral strategies againsthuman coronaviruses. Infect Disord Drug Targets 2007, 7:59-66.

13. Han TH, Chung JY, Kim SW, Hwang ES: Human Coronavirus-NL63 infections in Korean children, 2004–2006. J Clin Virol2007, 38:27-31.

14. Chiu SS, Chan KH, Chu KW, Kwan SW, Guan Y, Poon LL, Peiris JS:Human coronavirus NL63 infection and other coronavirusinfections in children hospitalized with acute respiratory dis-ease in Hong Kong, China. Clin Infect Dis 2005, 40:1721-1729.

15. Bastien N, Robinson JL, Tse A, Lee BE, Hart L, Li Y: Human coro-navirus NL-63 infections in children: a 1-year study. J ClinMicrobiol 2005, 43:4567-4573.

16. Ebihara T, Endo R, Ma X, Ishiguro N, Kikuta H: Detection ofhuman coronavirus NL63 in young children with bronchioli-tis. J Med Virol 2005, 75:463-465.

17. Moes E, Vijgen L, Keyaerts E, Zlateva K, Li S, Maes P, Pyrc K, BerkhoutB, Hoek L van der, Van Ranst M: A novel pancoronavirus RT-PCRassay: frequent detection of human coronavirus NL63 in chil-




































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dren hospitalized with respiratory tract infections in Bel-gium. BMC Infect Dis 2005, 5:6.

18. Pyrc K, Bosch BJ, Berkhout B, Jebbink MF, Dijkman R, Rottier P, HoekL van der: Inhibition of human coronavirus NL63 infection atearly stages of the replication cycle. Antimicrob Agents Chemother2006, 50:2000-2008.

19. Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL,Guan Y, Rozanov M, Spaan WJ, Gorbalenya AE: Unique and con-served features of genome and proteome of SARS-coronavi-rus, an early split-off from the coronavirus group 2 lineage. JMol Biol 2003, 331:991-1004.

20. Scandella E, Eriksson KK, Hertzig T, Drosten C, Chen L, Gui C, LuoX, Shen J, Shen X, Siddell SG, et al.: Identification and evaluationof coronavirus replicase inhibitors using a replicon cell line.Adv Exp Med Biol 2006, 581:609-613.

21. Chen L, Gui C, Luo X, Yang Q, Gunther S, Scandella E, Drosten C,Bai D, He X, Ludewig B, et al.: Cinanserin is an inhibitor of the3C-like proteinase of severe acute respiratory syndromecoronavirus and strongly reduces virus replication in vitro. JVirol 2005, 79:7095-7103.

22. Battegay M, Cooper S, Althage A, Banziger J, Hengartner H, Zinker-nagel RM: Quantification of lymphocytic choriomeningitisvirus with an immunological focus assay in 24- or 96-wellplates. J Virol Methods 1991, 33:191-198.

23. Matrosovich M, Matrosovich T, Garten W, Klenk HD: New low-vis-cosity overlay medium for viral plaque assays. Virol J 2006,3:63.

24. Leyssen P, Charlier N, Paeshuyse J, De Clercq E, Neyts J: Prospectsfor antiviral therapy. Adv Virus Res 2003, 61:511-553.

25. Leyssen P, De Clercq E, Neyts J: Molecular strategies to inhibitthe replication of RNA viruses. Antiviral Res 2008, 78:9-25.

26. Makino S, Taguchi F, Fujiwara K: Defective interfering particles ofmouse hepatitis virus. Virology 1984, 133:9-17.

27. Donaldson EF, Yount B, Sims AC, Burkett S, Pickles RJ, Baric RS: Sys-tematic Assembly of a Full-length Infectious Clone ofHuman Coronavirus NL63. J Virol 2008, 82(23):11948-11957.

28. Schildgen O, Jebbink MF, de Vries M, Pyrc K, Dijkman R, Simon A,Muller A, Kupfer B, Hoek L van der: Identification of cell linespermissive for human coronavirus NL63. J Virol Methods 2006,138:207-210.

29. Fouchier RA, Hartwig NG, Bestebroer TM, Niemeyer B, de Jong JC,Simon JH, Osterhaus AD: A previously undescribed coronavirusassociated with respiratory disease in humans. Proc Natl AcadSci USA 2004, 101:6212-6216.

30. Delmas B, Gelfi J, L'Haridon R, Vogel LK, Sjostrom H, Noren O,Laude H: Aminopeptidase N is a major receptor for theentero-pathogenic coronavirus TGEV. Nature 1992,357:417-420.

31. Reynolds DJ: Coronavirus replication in the intestinal and res-piratory tracts during infection of calves. Ann Rech Vet 1983,14:445-446.

32. Leung WK, To KF, Chan PK, Chan HL, Wu AK, Lee N, Yuen KY, SungJJ: Enteric involvement of severe acute respiratory syn-drome-associated coronavirus infection. Gastroenterology 2003,125:1011-1017.

33. Cinatl J Jr, Hoever G, Morgenstern B, Preiser W, Vogel JU, HofmannWK, Bauer G, Michaelis M, Rabenau HF, Doerr HW: Infection ofcultured intestinal epithelial cells with severe acute respira-tory syndrome coronavirus. Cell Mol Life Sci 2004, 61:2100-2112.

34. Yamashita M, Yamate M, Li GM, Ikuta K: Susceptibility of humanand rat neural cell lines to infection by SARS-coronavirus.Biochem Biophys Res Commun 2005, 334:79-85.

35. Hofmann H, Pyrc K, Hoek L van der, Geier M, Berkhout B, PohlmannS: Human coronavirus NL63 employs the severe acute respi-ratory syndrome coronavirus receptor for cellular entry.Proc Natl Acad Sci USA 2005, 102:7988-7993.

36. Hattermann K, Muller MA, Nitsche A, Wendt S, Donoso Mantke O,Niedrig M: Susceptibility of different eukaryotic cell lines toSARS-coronavirus. Arch Virol 2005, 150:1023-1031.

37. Weingartl HM, Copps J, Drebot MA, Marszal P, Smith G, Gren J,Andova M, Pasick J, Kitching P, Czub M: Susceptibility of pigs andchickens to SARS coronavirus. Emerg Infect Dis 2004,10:179-184.

38. de Souza Luna LK, Heiser V, Regamey N, Panning M, Drexler JF,Mulangu S, Poon L, Baumgarte S, Haijema BJ, Kaiser L, Drosten C:Generic detection of coronaviruses and differentiation at the

prototype strain level by RT-PCR and non-fluorescent lowdensity microarray. J Clin Microbiol 2007.


















































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